Fluorescence resonance energy transfer studies on anthrax lethal toxin

Area of Science:

• Bacterial Toxin Structure and Function
• Molecular Biology
• Biochemistry

Background:

• Anthrax lethal toxin comprises protective antigen (PA) and lethal factor (LF).
• PA(63) heptamers bind and translocate LF into eukaryotic cells.
• Understanding the interaction interface is crucial for toxin mechanism elucidation.

Purpose of the Study:

• To map the binding site of the lethal factor N-terminal domain (LF(N)) on the PA(63) heptamer.
• To investigate the structural organization of the assembled anthrax toxin complex.

Main Methods:

• Site-directed mutagenesis to introduce cysteine residues at specific positions on PA and LF(N).
• Fluorescent labeling of cysteine residues with IAEDANS and Alexa 488 maleimide.
• Time-resolved Förster Resonance Energy Transfer (FRET) to measure distances within the toxin complex.

Main Results:

• Fluorescence resonance energy transfer (FRET) was detected between PA residue N209C and LF(N).
• Minimal FRET was observed from PA residues E614C and E733C, indicating their distance from LF(N).
• Mutagenesis and labeling did not significantly impair toxin function.

Conclusions:

• LF(N) binds to the PA(63) heptamer in close proximity to residue N209.
• These findings support a model where LF(N) interacts near the top of the PA(63) heptamer ring.
• The study provides insights into the molecular assembly and interaction dynamics of anthrax lethal toxin.